The present invention relates to a method to selectively disrupt a particular organ, tissue, or cell of an animal at a specific period, and an expression unit, vector, host cell, and kit that are utilized in the method. The invention also relates to a non-human animal in which a particular organ, tissue, or cell is disrupted by the method.
A normal function of a tissue or cell can be clarified by deleting the tissue or cell and examining the resulting effect. This procedure is performed by, for example, surgical ablation of a tissue or cell (including laser ablation) and specific expression of a toxin in a target cell (Palmiter, R. D. et al. (1987) Cell 50, 435-443; Breitman, M. L. et al. (1987) Science 238, 1563-1565; Borrelli, E. et al. (1988) Proc. Natl. Acad. Sci. USA 85, 7572-7576; Heyman, R. A. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 2698-2702; Lowell, B. B. et al. (1993) Nature 3664 740-742; Ross, S. R. et al. (1993) Genes Dev. 7, 1318-1324).
When the target cells exist diffusely in the whole body, it is impossible to efficiently ablate only the target cells by the former method. When the target organ, tissue or cell is essential for normal development, the latter method results in death of the individual at embryo stage, and it is thus impossible to analyze animals after birth.
An objective of the present invention is to provide a method to specifically disrupt a particular organ, tissue, or cell at an arbitrary period.
Diphtheria toxin is a protein of 58 kDa and consists of two domains, fragment A and B. The fragment B domain binds to the precursor of heparin-binding EGF-like growth factor (HB-EGF)/diphtheria toxin receptor (DTR) which exists on the cell surface, and the toxin is incorporated into the cell via endocytosis. Upon fusion of the endosome with a lysosome, the conformation of the toxin is changed because of the effect of pH, and then the fragment A domain is translocated into the cytosol where the fragment inactivates peptide chain elongation factor (elongation factor 2) by ADP-ribosylating it and thus inhibits protein synthesis to bring the cell death (Honjo, T. et al. (1968) J. Biol. Chem. 243, 3553-3555; Pappenheimer, A. M. Jr. (1977) Annu. Rev. Biochem. 46, 69-94; Kohno, K. et al. (1986) Proc. Natl. Acad. Sci. USA 83, 4978-4982; Mekada, E. et al. (1988) J. Cell Biol. 107, 511-519; Moskaug, J. O. et al. (1991) J. Biol. Chem. 266, 2652-2659). The binding ability of diphtheria toxin to the precursor of HB-EGF differs among animal species. It is lower in mice than in humans or monkeys. Accordingly, a human and a monkey are sensitive to the toxin, whereas mice are not sensitive (Mitamura, T. et al. (1995) J. Biol. Chem. 270 (3), 1015-1019). Therefore, if a transgenic mouse is produced by introducing the human HB-EGF (hHB-EGF)/DTR gene inserted at the downstream of a promoter that functions specifically in a target organ, tissue, or cell so as to express the gene specifically there, the target can be disrupted at an arbitrary period by administrating diphtheria toxin to the mouse.
In fact, it has been shown that L cells, a mouse fibroblast insensitive to diphtheria toxin, become sensitive to the toxin when the hHB-EGF/DTR gene was introduced and expressed (Naglish, J. G. et al. (1992) Cell 69, 1051-1061).
The present inventors selected the enhancer/promoter of the albumin gene, which is specifically expressed in hepatic parenchymal cells (Gorski, K. et al. (1986) Cell 47, 767-776; Pinkert, C. A. et al. (1987) Genes Dev. 1, 268-276) as a specific promoter for a particular organ, tissue, or cell. The hHB-EGF/DTR gene was ligated to the downstream of the promoter, and transgenic mice were created by introducing the expression unit. Next, the inventors confirmed that the hHB-EGF/DTR gene was actually expressed specifically in the hepatic cells of the transgenic mice and examined the process of the disruption of hepatic parenchymal cells by administrating diphtheria toxin to the mice. As a result, the inventors found hepatic cells of the transgenic mice were specifically disrupted depending on the administration period of the toxin, and accomplished the present invention.
Thus, the present invention relates to a method for specifically disrupting a desired organ, tissue, or cell at a desired period by administrating a compound, and more specifically relates to:
(1) an expression unit comprising a gene encoding a receptor for a compound, which is essentially nontoxic to a host, at the downstream of a promoter functioning specifically in a particular organ, tissue, or cell, wherein said compound binds to said receptor artificially expressed in the host and shows a selective toxicity to the cell expressing said receptor;
(2) the expression unit according to (1), wherein said compound is diphtheria toxin, and said receptor for the toxin is the precursor of heparin-binding EGF-like growth factor (HB-EGF)/diphtheria toxin receptor;
(3) a vector comprising the expression unit of (1) or (2);
(4) a host cell carrying the expression unit of (1) or (2), or a vector of (3);
(5) a non-human animal carrying the expression unit of (1) or (2), or the vector of (3);
(6) a method for specifically disrupting a particular organ, tissue, or cell at a particular period in the animal of (5), wherein said method comprises administering a compound that is essentially nontoxic to the host at said particular period;
(7) the method of (6), wherein said animal is a mouse, and said compound is diphtheria toxin;
(8) a non-human animal, in which a particular organ, tissue, or cell is disrupted by the method of (6) or (7);
(9) a kit comprising the compound of (1) and a DNA comprising the expression unit of (1).
The invention relates to an expression unit comprising a DNA encoding a receptor for a compound, which is essentially nontoxic to the host, at the downstream of a promoter functioning specifically in a particular organ, tissue, or cell.
The promoter functioning specifically in a particular organ, tissue, or cell is not particularly limited. Examples of the promoter includes those of genes: the H-2 class II gene, specific for macrophages and dendritic cells; interferon xcex1, for granulocytes; interleukins 2, 4, and 5, for T lymphocytes; insulin, for xcex2 cells in pancreas; GDNF, for glia cells; immunoglobulin, for B lymphocytes; BDNF, for neurons; HGF, for hepatic parenchymal cells; THY-1, for T lymphocytes and neurons; T-cell receptor, for T lymphocytes; and hydroxymethylglutaryl coenzyme A reductase (HMG) and factor IX, for liver.
For example, the compound that is essentially nontoxic to a host includes diphtheria toxin when the host is a mouse. The compound is not particularly limited as long as it has no toxicity in hosts into which a receptor gene is not introduced, but has a selective toxicity to a particular organ, tissue, or cell of the hosts into which a receptor gene is introduced and expressed.
The receptor for a compound that is essentially nontoxic to the host is not particularly limited as long as it exhibits toxicity to a cell expressing the receptor by binding to the compound. However, a receptor whose expression itself has a bad influence on the cell is not preferable. When the compound is diphtheria toxin, a preferable receptor is the precursor of HB-EGF (heparin-binding EGF-like growth factor)/diphtheria toxin receptor.
A form of a DNA encoding the receptor is not limited as long as it encodes the receptor protein. The DNA includes cDNA, genomic DNA, and chemically synthesized DNA, etc. The DNA encoding the receptor can be prepared by a standard method (Molecular Cloning, 2nd edition, chapters 16 and 17, Cold Spring Harbor Laboratory Press).
The DNA can be inserted at the downstream of the promoter by a standard method (Molecular Cloning, 2nd edition, chapters 16 and 17, Cold Spring Harbor Laboratory Press). The resulting product can be an expression unit of the invention.
The invention also relates to a vector comprising the above expression unit.
The vector of the present invention is not particularly limited as long as it carries a gene capable of expression in eucaryotes. The expression plasmid vector can be introduced into substantially any hosts by, for example, calcium phosphate method, DEAE-dextran method, lipofection, liposome method, HVJ membrane fusion liposome method, microinjection, particle gun technique, and electroporation. In addition, a variety of retrovirus vectors which have host-selective infectivity can be utilized. Adenovirus vector, adeno-associated virus vector, herpes virus vector, HIV vector, and sindbis virus vector are also utilized.
The expression unit can be introduced into the vector of the present invention by treating a desired nucleic acid fragment with a desired restriction enzyme to make a cohesive end at a unique restriction site and by ligating the fragment with a compatible vector fragment (Molecular Cloning, 2nd edition, chapters 16 and 17, Cold Spring Harbor Laboratory Press).
The invention also relates to a host cell carrying the above vector.
A variety of animal cells can be used, without limitation, as a host cell, into which the vector of the present invention is introduced.
The vector can be introduced into host cells by, for example, chemical methods such as calcium phosphate method (Molecular Cloning, 16.32, Cold Spring Harbor Laboratory Press (1989)), DEAE-dextran method (Molecular Cloning, 16.41, Cold Spring Harbor Laboratory Press (1989)), lipofection (Annu. N.Y. Acad. Sci. 716, 23-34 (1994)), and liposome method (Annu. N.Y. Acad. Sci. 716, 144-153 (1994)), for example. In addition, a gene carrier system utilizing polycations, such as polylysine, conjugated with a protein or sugar has been recently studied (J. Controlled Release 19, 269-274 (1992)). Physical methods can also be used and include microinjection, by which a nucleic acid is directly injected into cells (Hypertension 22, 599-607 (1993)); particle gun technique, by which a gold particle covered with a nucleic acid is shot through target cells (Proc. Natl. Acad. Sci. USA 87, 9568 (1990)); and electroporation (Cancer Treat Rev. 20, 105-115 (1994)). However, any of the above methods is incapable of integrating an introduced gene into chromosomes, and it is thus difficult to obtain long-term retaining or expression of the gene. Preferably, a vector is prepared to contain an appropriate element for retaining a nucleic acid in host cells for sufficient time so that the nucleic acid can be inserted into a desired site. Such a vector is able to enter into higher eucaryotic cells, and preferably to be integrated into chromosomes. A vector which is contained as an extrachromosomal DNA can also be used as long as it has ability to express a receptor-encoding foreign gene for a desired period. Specific examples of the vector are recombinant virus vectors, including those derived from adenovirus, retroviruses, and adeno-associated virus. Alternatively, a foreign gene can be introduced into a desired chromosomal site or nucleic acid region by using integrase and a protein that recognizes a specific DNA sequence (Science 267, 1443-1444 (1995)).
Moreover, the present invention relates to a non-human animal, carrying the above expression unit or vector, and a method for disrupting a particular organ in the animal by administering a compound. The animal of the invention is characterized by specifically expressing a receptor for the above compound in a particular organ, tissue, or cell depending on the function of an introduced promoter. Therefore, when the compound is administered into a non-human animal of the invention, the compound showed toxicity in a particular organ, tissue, or cell through binding to a receptor that is specifically expressed in the particular site. It is thus possible to specifically disrupt a particular organ in the animal depending on the administration period of the compound. The administration period is not particularly limited, but is preferably a period when the compound concentration in blood sufficiently exceeds the half saturation concentration of a receptor for a compound that is essentially nontoxic to the hosts.
The non-human animal can be generated by a known method for generating transgenic animals. For example, the method includes a method of directly injecting a gene into the pronucleus of a fertilized egg using a micropipet under a phase contrast microscopy (microinjection; Proc. Natl. Acad. Sci. USA 77, 7380-7384 (1980)), a method using a recombinant retrovirus vector (retrovirus method; Proc. Natl. Acad. Sci. USA 82, 6148-6152, 6927-6931, 8587-8591 (1985)), and a method using an embryonic stem cell (ES cell) (Proc. Natl. Acad. Sci. USA 83, 9065-9069 (1986)). The non-human animal of the present invention is basically generated by a method of generating transgenic mice, which includes three known methods (Hogan, B. et al. A manual for mouse embryo manipulation, Kindai Shuppan, Tokyo (1989); Nomura, T. et al. A laboratory manual for developmental technology, Kodansya, Tokyo (1987); and Yamamura, K. Molecular Medicine in Mice, Nankodo Co., Ltd., Tokyo (1993)). Specifically, transgenic mice can be generated by, for example, harvesting fertilized eggs, injecting an isolated gene into the pronuclei of the eggs using a micromanipulator, and then transplanting the obtained eggs into a uterine tube. Alternatively, transgenic mice can be generated by introducing a desired gene into an ES clone by electroporation, selecting desired cells in terms of drug resistance, injecting the selected cells into fertilized eggs using a micromanipulator to create chimera embryos.
Because of tissue- and cell-specific function of the gene promoter, the generated non-human animal can be used as a pathological model of a disease originating from a particular cell, specifically a model animal of diabetes or neuropathy. It can also be used as a model animal for studying the mechanism of memory by depleting neurons involved in memory at a particular developmental stage. In addition, minituarized animals can be generated by arresting the animal growth at a desired period.
Furthermore, the present invention relates to a kit comprising a compound essentially nontoxic to hosts and a DNA comprising an expression unit comprising a DNA encoding a receptor for the compound at the downstream of a promoter functioning specifically in a particular organ, tissue, or cell. For example, xe2x80x9ca DNA comprising an expression unitxe2x80x9d contained in the kit of the invention is introduced into animal cells to create a transgenic animal, and xe2x80x9ca compound essentially nontoxic to a hostxe2x80x9d that is also contained in the kit of the invention is administered to the transgenic animal at a desired time period, that a particular organ of the transgenic animal can be specifically disrupted depending on the administration period of the compound.